Transport for All, which campaigns on behalf of disabled transport users, took aim at the beleaguered operator in Wednesday evening’s protest.
Southern has been embroiled in a year-long dispute with its staff over changes to the role of guards as well as driver-only operated trains.
Transport for All said previous train maps specified the stations where passengers needing assistance could turn up and travel.
Now, the maps on the trains say that if such passengers do not book help in advance, "there might be a significant delay to your journey".
More than 50 disabled people and their supporters turned up at the busy central London station for the protest.
One protester, Paula Peters told the Standard: “We have to book a time to travel and return home. We cannot travel as non-disabled people travel and turn up and go.
“We want rail access now and fully accessible rail travel so we can live our lives.”
Protesters began by marching to London Bridge and then handed out petition cards to commuters making their way home station. It ended with speakers discussing why rail access is important for disabled people.
Transport for All said it hears daily of the "unacceptable treatment" faced by many disabled and older travellers on the railways.
Faryal Velmi, director of Transport for All, said: "It's shameful that Southern Rail is allowing disabled passengers to bear the brunt of their failures to deliver a reliable service.
"We have heard daily from disabled transport users stranded on freezing platforms or forced to crawl onto trains when rail companies have failed to assist them."
A Southern spokesman said before the protest: "Passengers do not have to book assistance before travelling with us.
"We only recommend this to ensure we have staff prepared with ramps or that alternative travel is in place if a station is not accessible.
"We now have more staff assigned to work on our trains than we did before who can assist our passengers.
"Any trains that do run without an on-board supervisor do so only under exceptional circumstances and previously that train would have had to be cancelled, inconveniencing not just the passenger in need of boarding assistance but hundreds of other passengers as well.
"Our priority is to have an on-board supervisor on services which previously had a conductor and in the exceptional circumstances when this is not possible, we have a clear, robust process to ensure passengers with accessibility requirements are assisted to complete their journeys."